Transgenic mice containing DEZ orphan receptor gene disruptions

ABSTRACT

The present invention relates to transgenic animals, as well as compositions and methods relating to the characterization of gene function. Specifically, the present invention provides transgenic mice comprising mutations in a DEZ receptor gene. Such transgenic mice are useful as models for disease and for identifying agents that modulate gene expression and gene function, and as potential treatments for various disease states and disease conditions.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/216,253, filed Jul. 6, 2000; U.S. Provisional Application No.60/219,403, filed Jul. 19, 2000; U.S. Provisional Application No.60/251,815, filed Dec. 6, 2000; and U.S. Provisional Application No.60/262,137, filed Jan. 16, 2001, the entire contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to transgenic animals, compositionsand methods relating to the characterization of gene function.

BACKGROUND OF THE INVENTION

[0003] Many medically significant biological processes are mediated byproteins participating in signal transduction pathways that involveG-proteins and/or second messengers such as cAMP. The membrane proteingene superfamily of G-protein coupled receptors (GPCRs) includes a widerange of biologically active receptors, such as hormone, viral, growthfactor and neuroreceptors. GPCRs have been characterized as having sevenputative transmembrane domains (designated TM1, TM2, TM3, TM4, TM5, TM6,and TM7), which are believed to represent transmembrane α-helicesconnected by extracellular or cytoplasmic loops. Most G-protein coupledreceptors have single conserved cysteine residues in each of the firsttwo extracellular loops that form disulfide bonds that are believed tostabilize functional protein structure. G-protein coupled receptors canbe intracellularly coupled by heterotrimeric G-proteins to variousintracellular enzymes, ion channels and transporters. DifferentG-protein α-subunits preferentially stimulate particular effectors tomodulate various biological functions in a cell.

[0004] Recently, a novel orphan GPCR termed the DEZ receptor (akachemokine-like receptor 1, or CMKLR1) was isolated from a cDNA derivedfrom the cell line NH15-CA2 and a cDNA library from adult mouse brainusing a PCR cloning strategy (Methner et al., Biochem. Biophys. Res.Commun. 233(2): 336-342 (1997)). The amino acid sequence of this DEZorphan receptor showed homology to neuropeptide and chemoattractantreceptors. Highest overall homology was found with the orphan receptorGPR-1 (65%), the angiotensin II receptor (62%), and the C5aanaphylatoxin receptor (60%). In situ hybridization experiments showedthat dez is differentially regulated during development, with aprominent expression in developing osseous and cartilaginous tissue. Itwas also detectable in the adult parathyroid glands, hinting at apossible function in bone metabolism. However, the structural andfunctional attributes of this receptor have yet to be determined.

[0005] The complete 1892 bp cds for the murine DEZ orphan receptor, orCMKLR1, has been deposited in GenBank (Accession number: U79525; GI/NIDnumber: 1732346).

[0006] Given the importance of GPCRs, particularly the DEZ orphanreceptor, a clear need exists for identification and characterization ofGPCRs which can play a role in preventing, ameliorating or correctingdysfunctions or diseases.

SUMMARY OF THE INVENTION

[0007] The present invention generally relates to transgenic animals, aswell as to compositions and methods relating to the characterization ofgene function.

[0008] The present invention provides transgenic cells comprising adisruption in a DEZ receptor gene. The transgenic cells of the presentinvention are comprised of any cells capable of undergoing homologousrecombination. Preferably, the cells of the present invention are stemcells and more preferably, embryonic stem (ES) cells, and mostpreferably, murine ES cells. According to one embodiment, the transgeniccells are produced by introducing a targeting construct into a stem cellto produce a homologous recombinant, resulting in a mutation of the DEZreceptor gene. In another embodiment, the transgenic cells are derivedfrom the transgenic animals described below. The cells derived from thetransgenic animals includes cells that are isolated or present in atissue or organ, and any cell lines or any progeny thereof.

[0009] The present invention also provides a targeting construct andmethods of producing the targeting construct that when introduced intostem cells produces a homologous recombinant. In one embodiment, thetargeting construct of the present invention comprises first and secondpolynucleotide sequences that are homologous to the DEZ receptor gene.The targeting construct also comprises a polynucleotide sequence thatencodes a selectable marker that is preferably positioned between thetwo different homologous polynucleotide sequences in the construct. Thetargeting construct may also comprise other regulatory elements that mayenhance homologous recombination.

[0010] The present invention further provides non-human transgenicanimals and methods of producing such non-human transgenic animalscomprising a disruption in a DEZ receptor gene. The transgenic animalsof the present invention include transgenic animals that areheterozygous and homozygous for a mutation in the DEZ receptor gene. Inone aspect, the transgenic animals of the present invention aredefective in the function of the DEZ receptor gene. In another aspect,the transgenic animals of the present invention comprise a phenotypeassociated with having a mutation in a DEZ receptor gene.

[0011] The present invention also provides methods of identifying agentscapable of affecting a phenotype of a transgenic animal. For example, aputative agent is administered to the transgenic animal and a responseof the transgenic animal to the putative agent is measured and comparedto the response of a “normal” or wild type mouse, or alternativelycompared to a transgenic animal control (without agent administration).The invention further provides agents identified according to suchmethods. The present invention also provides methods of identifyingagents useful as therapeutic agents for treating conditions associatedwith a disruption of the DEZ receptor gene.

[0012] The present invention further provides a method of identifyingagents having an effect on DEZ receptor expression or function. Themethod includes administering an effective amount of the agent to atransgenic animal, preferably a mouse. The method includes measuring aresponse of the transgenic animal, for example, to the agent, andcomparing the response of the transgenic animal to a control animal,which may be, for example, a wild-type animal or alternatively, atransgenic animal control. Compounds that may have an effect on DEZreceptor expression or function may also be screened against cells incell-based assays, for example, to identify such compounds.

[0013] The invention also provides cell lines comprising nucleic acidsequences of a DEZ receptor gene. Such cell lines may be capable ofexpressing such sequences by virtue of operable linkage to a promoterfunctional in the cell line. Preferably, expression of the DEZ receptorgene sequence is under the control of an inducible promoter. Alsoprovided are methods of identifying agents that interact with the DEZreceptor gene, comprising the steps of contacting the DEZ receptor genewith an agent and detecting an agent/DEZ receptor gene complex. Suchcomplexes can be detected by, for example, measuring expression of anoperably linked detectable marker.

[0014] The invention further provides methods of treating diseases orconditions associated with a disruption in a DEZ receptor gene, and moreparticularly, to a disruption in the expression or function of the DEZreceptor gene. In a preferred embodiment, methods of the presentinvention involve treating diseases or conditions associated with adisruption in the DEZ receptor gene's expression or function, includingadministering to a subject in need, a therapeutic agent that effects DEZreceptor expression or function. In accordance with this embodiment, themethod comprises administration of a therapeutically effective amount ofa natural, synthetic, semi-synthetic, or recombinant DEZ receptor gene,DEZ receptor gene products or fragments thereof as well as natural,synthetic, semi-synthetic or recombinant analogs.

[0015] The present invention further provides methods of treatingdiseases or conditions associated with disrupted targeted geneexpression or function, wherein the methods comprise detecting andreplacing through gene therapy mutated DEZ receptor genes.

[0016] Definitions

[0017] The term “gene” refers to (a) a gene containing at least one ofthe DNA sequences disclosed herein; (b) any DNA sequence that encodesthe amino acid sequence encoded by the DNA sequences disclosed hereinand/or; (c) any DNA sequence that hybridizes to the complement of thecoding sequences disclosed herein. Preferably, the term includes codingas well as noncoding regions, and preferably includes all sequencesnecessary for normal gene expression including promoters, enhancers andother regulatory sequences.

[0018] The terms “polynucleotide” and “nucleic acid molecule” are usedinterchangeably to refer to polymeric forms of nucleotides of anylength. The polynucleotides may contain deoxyribonucleotides,ribonucleotides and/or their analogs. Nucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The term “polynucleotide” includes single-, double-stranded andtriple helical molecules. “Oligonucleotide” refers to polynucleotides ofbetween 5 and about 100 nucleotides of single- or double-stranded DNA.Oligonucleotides are also known as oligomers or oligos and may beisolated from genes, or chemically synthesized by methods known in theart. A “primer” refers to an oligonucleotide, usually single-stranded,that provides a 3′-hydroxyl end for the initiation of enzyme-mediatednucleic acid synthesis. The following are non-limiting embodiments ofpolynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA,rRNA, ribozymes, cDNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence,isolated RNA of any sequence, nucleic acid probes and primers. A nucleicacid molecule may also comprise modified nucleic acid molecules, such asmethylated nucleic acid molecules and nucleic acid molecule analogs.Analogs of purines and pyrimidines are known in the art, and include,but are not limited to, aziridinycytosine, 4-acetylcytosine,5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine,1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, pseudouracil, 5-pentylnyluracil and 2,6-diaminopurine.The use of uracil as a substitute for thymine in a deoxyribonucleic acidis also considered an analogous form of pyrimidine.

[0019] A “fragment” of a polynucleotide is a polynucleotide comprised ofat least 9 contiguous nucleotides, preferably at least 15 contiguousnucleotides and more preferably at least 45 nucleotides, of coding ornon-coding sequences.

[0020] The term “gene targeting” refers to a type of homologousrecombination that occurs when a fragment of genomic DNA is introducedinto a mammalian cell and that fragment locates and recombines withendogenous homologous sequences.

[0021] The term “homologous recombination” refers to the exchange of DNAfragments between two DNA molecules or chromatids at the site ofhomologous nucleotide sequences.

[0022] The term “homologous” as used herein denotes a characteristic ofa DNA sequence having at least about 70 percent sequence identity ascompared to a reference sequence, typically at least about 85 percentsequence identity, preferably at least about 95 percent sequenceidentity, and more preferably about 98 percent sequence identity, andmost preferably about 100 percent sequence identity as compared to areference sequence. Homology can be determined using a “BLASTN”algorithm. It is understood that homologous sequences can accommodateinsertions, deletions and substitutions in the nucleotide sequence.Thus, linear sequences of nucleotides can be essentially identical evenif some of the nucleotide residues do not precisely correspond or align.The reference sequence may be a subset of a larger sequence, such as aportion of a gene or flanking sequence, or a repetitive portion of achromosome.

[0023] The term “target gene” (alternatively referred to as “target genesequence” or “target DNA sequence” or “target sequence”) refers to anynucleic acid molecule or polynucleotide of any gene to be modified byhomologous recombination. The target sequence includes an intact gene,an exon or intron, a regulatory sequence or any region between genes.The target gene comprises a portion of a particular gene or geneticlocus in the individual's genomic DNA. As provided herein, the targetgene of the present invention is a DEZ receptor (or CMKLR1) gene. A “DEZreceptor gene” as used herein refers to a sequence comprising SEQ IDNO:1, i.e., comprising the sequence identified in GenBank as Accessionnumber: U79525; GI/NID number: 1732346, which is also referred to as a“CMKLR1 gene.”

[0024] “Disruption” of a DEZ receptor gene occurs when a fragment ofgenomic DNA locates and recombines with an endogenous homologoussequence. These sequence disruptions or modifications may includeinsertions, missense, frameshift, deletion, or substitutions, orreplacements of DNA sequence, or any combination thereof. Insertionsinclude the insertion of entire genes, which may be of animal, plant,fungal, insect, prokaryotic, or viral origin. Disruption, for example,can alter or replace a promoter, enhancer, or splice site of a DEZreceptor gene, and can alter the normal gene product by inhibiting itsproduction partially or completely or by enhancing the normal geneproduct's activity.

[0025] The term, “transgenic cell”, refers to a cell containing withinits genome a DEZ receptor gene that has been disrupted, modified,altered, or replaced completely or partially by the method of genetargeting.

[0026] The term “transgenic animal” refers to an animal that containswithin its genome a specific gene that has been disrupted by the methodof gene targeting. The transgenic animal includes both the heterozygoteanimal (i.e., one defective allele and one wild-type allele) and thehomozygous animal (i.e., two defective alleles).

[0027] As used herein, the terms “selectable marker” or “positiveselection marker” refers to a gene encoding a product that enables onlythe cells that carry the gene to survive and/or grow under certainconditions. For example, plant and animal cells that express theintroduced neomycin resistance (Neo^(r)) gene are resistant to thecompound G418. Cells that do not carry the Neo^(r) gene marker arekilled by G418. Other positive selection markers will be known to thoseof skill in the art.

[0028] A “host cell” includes an individual cell or cell culture thatcan be or has been a recipient for vector(s) or for incorporation ofnucleic acid molecules and/or proteins. Host cells include progeny of asingle host cell, and the progeny may not necessarily be completelyidentical (in morphology or in total DNA complement) to the originalparent due to natural, accidental, or deliberate mutation. A host cellincludes cells transfected with the constructs of the present invention.

[0029] The term “modulates” as used herein refers to the inhibition,reduction, increase or enhancement of a DEZ receptor function,expression, activity, or alternatively a phenotype associated with adisruption in a DEZ receptor gene. In a preferred embodiment, the term“modulates” refers to agents that act as agonists or antagonists of thereceptor.

[0030] The term “ameliorates” refers to a decreasing, reducing oreliminating of a condition, disease, disorder, or phenotype, includingan abnormality or symptom associated with a disruption in a DEZ receptorgene.

[0031] The term “abnormality” refers to any disease, disorder,condition, or phenotype in which a disruption of a DEZ receptor gene isimplicated, including pathological conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 shows a polynucleotide sequence for the target gene, a DEZreceptor, or CMKLR1 (SEQ ID NO:1), identified as GenBank Accessionnumber: U79525; GI/NID number: 1732346.

[0033] FIGS. 2A-2B show design of the targeting construct used todisrupt DEZ receptor genes. FIG. 2B shows the sequences identified asSEQ ID NO:2 and SEQ ID NO:3, which were used as the targeting arms(homologous sequences) in the DEZ receptor targeting construct.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The invention is based, in part, on the evaluation of theexpression and role of genes and gene expression products, primarilythose associated with a DEZ receptor. Among others, the inventionpermits the definition of disease pathways and the identification ofdiagnostically and therapeutically useful targets. For example, genesthat are mutated or down-regulated under disease conditions may beinvolved in causing or exacerbating the disease condition. Treatmentsdirected at up-regulating the activity of such genes or treatments thatinvolve alternate pathways, may ameliorate the disease condition.

[0035] Generation of Targeting Construct

[0036] The targeting construct of the present invention may be producedusing standard methods known in the art. (see, e.g., Sambrook, et al.,1989, Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; E. N. Glover(eds.), 1985, DNA Cloning: A Practical Approach, Volumes I and II; M. J.Gait (ed.), 1984, Oligonucleotide Synthesis; B. D. Hames & S. J. Higgins(eds.), 1985, Nucleic Acid Hybridization; B. D. Hames & S. J. Higgins(eds.), 1984, Transcription and Translation; R. I. Freshney (ed.), 1986,Animal Cell Culture; Immobilized Cells and Enzymes, IRL Press, 1986; B.Perbal, 1984, A Practical Guide To Molecular Cloning; F. M. Ausubel etal., 1994, Current Protocols in Molecular Biology, John Wiley & Sons,Inc.). For example, the targeting construct may be prepared inaccordance with conventional ways, where sequences may be synthesized,isolated from natural sources, manipulated, cloned, ligated, subjectedto in vitro mutagenesis, primer repair, or the like. At various stages,the joined sequences may be cloned, and analyzed by restrictionanalysis, sequencing, or the like.

[0037] The targeting DNA can be constructed using techniques well knownin the art. For example, the targeting DNA may be produced by chemicalsynthesis of oligonucleotides, nick-translation of a double-stranded DNAtemplate, polymerase chain-reaction amplification of a sequence (orligase chain reaction amplification), purification of prokaryotic ortarget cloning vectors harboring a sequence of interest (e.g., a clonedcDNA or genomic DNA, synthetic DNA or from any of the aforementionedcombination) such as plasmids, phagemids, YACs, cosmids, bacteriophageDNA, other viral DNA or replication intermediates, or purifiedrestriction fragments thereof, as well as other sources of single anddouble-stranded polynucleotides having a desired nucleotide sequence.Moreover, the length of homology may be selected using known methods inthe art. For example, selection may be based on the sequence compositionand complexity of the predetermined endogenous target DNA sequence(s).

[0038] The targeting construct of the present invention typicallycomprises a first sequence homologous to a portion or region of the DEZreceptor gene and a second sequence homologous to a second portion orregion of the DEZ receptor gene. The targeting construct furthercomprises a positive selection marker, which is preferably positioned inbetween the first and the second DNA sequence that are homologous to aportion or region of the target DNA sequence. The positive selectionmarker may be operatively linked to a promoter and a polyadenylationsignal.

[0039] Other regulatory sequences known in the art may be incorporatedinto the targeting construct to disrupt or control expression of aparticular gene in a specific cell type. In addition, the targetingconstruct may also include a sequence coding for a screening marker, forexample, green fluorescent protein (GFP), or another modifiedfluorescent protein.

[0040] Although the size of the homologous sequence is not critical andcan range from as few as 50 base pairs to as many as 100 kb, preferablyeach fragment is greater than about 1 kb in length, more preferablybetween about 1 and about 10 kb, and even more preferably between about1 and about 5 kb. One of skill in the art will recognize that althoughlarger fragments may increase the number of homologous recombinationevents in ES cells, larger fragments will also be more difficult toclone.

[0041] In a preferred embodiment of the present invention, the targetingconstruct is prepared directly from a plasmid genomic library using themethods described in pending U.S. patent application Ser. No.:08/971,310, filed Nov. 17, 1997, the disclosure of which is incorporatedherein in its entirety. Generally, a sequence of interest is identifiedand isolated from a plasmid library in a single step using, for example,long-range PCR. Following isolation of this sequence, a secondpolynucleotide that will disrupt the target sequence can be readilyinserted between two regions encoding the sequence of interest. Inaccordance with this aspect, the construct is generated in two steps by(1) amplifying (for example, using long-range PCR) sequences homologousto the target sequence, and (2) inserting another polynucleotide (forexample a selectable marker) into the PCR product so that it is flankedby the homologous sequences. Typically, the vector is a plasmid from aplasmid genomic library. The completed construct is also typically acircular plasmid.

[0042] In another embodiment, the targeting construct is designed inaccordance with the regulated positive selection method described inU.S. patent application Ser. No. 60/232,957, filed Sep. 15, 2000, thedisclosure of which is incorporated herein in its entirety. Thetargeting construct is designed to include a PGK-neo fusion gene havingtwo lacO sites, positioned in the PGK promoter and an NLS-lacI genecomprising a lac repressor fused to sequences encoding the NLS from theSV40 T antigen.

[0043] In another embodiment, the targeting construct may contain morethan one selectable maker gene, including a negative selectable marker,such as the herpes simplex virus tk (HSV-tk) gene. The negativeselectable marker may be operatively linked to a promoter and apolyadenylation signal. (see, e.g., U.S. Pat. Nos. 5,464,764; 5,487,992;5,627,059; and 5,631,153).

[0044] Generation of Cells and Confirmation of Homologous RecombinationEvents

[0045] Once an appropriate targeting construct has been prepared, thetargeting construct may be introduced into an appropriate host cellusing any method known in the art. Various techniques may be employed inthe present invention, including, for example, pronuclearmicroinjection; retrovirus mediated gene transfer into germ lines; genetargeting in embryonic stem cells; electroporation of embryos;sperm-mediated gene transfer; and calcium phosphate/DNA co-precipitates,microinjection of DNA into the nucleus, bacterial protoplast fusion withintact cells, transfection, polycations, e.g., polybrene, polyomithine,etc., or the like (see, e.g., U.S. Pat. No. 4,873,191; Van der Putten,et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152; Thompson, etal., 1989, Cell 56:313-321; Lo, 1983, Mol Cell. Biol. 3:1803-1814;Lavitrano, et al., 1989, Cell, 57:717-723). Various techniques fortransforming mammalian cells are known in the art. (see, e.g., Gordon,1989, Intl. Rev. Cytol., 115:171-229; Keown et al., 1989, Methods inEnzymology; Keown et al., 1990, Methods and Enzymology, Vol. 185, pp.527-537; Mansour et al., 1988, Nature, 336:348-352).

[0046] In a preferred aspect of the present invention, the targetingconstruct is introduced into host cells by electroporation. In thisprocess, electrical impulses of high field strength reversiblypermeabilize biomembranes allowing the introduction of the construct.The pores created during electroporation permit the uptake ofmacromolecules such as DNA. (see, e.g., Potter, H., et al., 1984, Proc.Nat'l. Acad. Sci. U.S.A. 81:7161-7165).

[0047] Any cell type capable of homologous recombination may be used inthe practice of the present invention. Examples of such target cellsinclude cells derived from vertebrates including mammals such as humans,bovine species, ovine species, murine species, simian species, and ethereucaryotic organisms such as filamentous fungi, and higher multicellularorganisms such as plants.

[0048] Preferred cell types include embryonic stem (ES) cells, which aretypically obtained from pre-implantation embryos cultured in vitro.(see, e.g., Evans, M. J., et al., 1981, Nature 292:154-156; Bradley, M.O., et al., 1984, Nature 309:255-258; Gossler et al., 1986, Proc. Natl.Acad. Sci. USA 83:9065-9069; and Robertson, et al., 1986, Nature322:445-448). The ES cells are cultured and prepared for introduction ofthe targeting construct using methods well known to the skilled artisan.(see, e.g., Robertson, E. J. ed. “Teratocarcinomas and Embryonic StemCells, a Practical Approach”, IRL Press, Washington D.C., 1987; Bradleyet al., 1986, Current Topics in Devel. Biol. 20:357-371; by Hogan et al.in “Manipulating the Mouse Embryo”: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor N.Y., 1986; Thomas et al.,1987, Cell 51:503; Koller et al., 1991, Proc. Natl. Acad. Sci. USA,88:10730; Dorin et al., 1992, Transgenic Res. 1:101; and Veis et al.,1993, Cell 75:229). The ES cells that will be inserted with thetargeting construct are derived from an embryo or blastocyst of the samespecies as the developing embryo into which they are to be introduced.ES cells are typically selected for their ability to integrate into theinner cell mass and contribute to the germ line of an individual whenintroduced into the mammal in an embryo at the blastocyst stage ofdevelopment. Thus, any ES cell line having this capability is suitablefor use in the practice of the present invention.

[0049] The present invention may also be used to knockout genes in othercell types, such as stem cells. By way of example, stem cells may bemyeloid, lymphoid, or neural progenitor and precursor cells. These cellscomprising a disruption or knockout of a gene may be particularly usefulin the study of DEZ receptor gene function in individual developmentalpathways. Stem cells may be derived from any vertebrate species, such asmouse, rat, dog, cat, pig, rabbit, human, non-human primates and thelike.

[0050] After the targeting construct has been introduced into cells, thecells where successful gene targeting has occurred are identified.Insertion of the targeting construct into the targeted gene is typicallydetected by identifying cells for expression of the marker gene. In apreferred embodiment, the cells transformed with the targeting constructof the present invention are subjected to treatment with an appropriateagent that selects against cells not expressing the selectable marker.Only those cells expressing the selectable marker gene survive and/orgrow under certain conditions. For example, cells that express theintroduced neomycin resistance gene are resistant to the compound G418,while cells that do not express the neo gene marker are killed by G418.If the targeting construct also comprises a screening marker such asGFP, homologous recombination can be identified through screening cellcolonies under a fluorescent light. Cells that have undergone homologousrecombination will have deleted the GFP gene and will not fluoresce.

[0051] If a regulated positive selection method is used in identifyinghomologous recombination events, the targeting construct is designed sothat the expression of the selectable marker gene is regulated in amanner such that expression is inhibited following random integrationbut is permitted (derepressed) following homologous recombination. Moreparticularly, the transfected cells are screened for expression of theneo gene, which requires that (1) the cell was successfullyelectroporated, and (2) lac repressor inhibition of neo transcriptionwas relieved by homologous recombination. This method allows for theidentification of transfected cells and homologous recombinants to occurin one step with the addition of a single drug.

[0052] Alternatively, a positive-negative selection technique may beused to select homologous recombinants. This technique involves aprocess in which a first drug is added to the cell population, forexample, a neomycin-like drug to select for growth of transfected cells,i.e. positive selection. A second drug, such as FIAU is subsequentlyadded to kill cells that express the negative selection marker, i.e.negative selection. Cells that contain and express the negativeselection marker are killed by a selecting agent, whereas cells that donot contain and express the negative selection marker survive. Forexample, cells with non-homologous insertion of the construct expressHSV thymidine kinase and therefore are sensitive to the herpes drugssuch as gancyclovir (GANC) or FIAU (1-(2-deoxy2-fluoro-B-D-arabinofluranosyl)-5-iodouracil). (see, e.g., Mansour etal., Nature 336:348-352: (1988); Capecchi, Science 244:1288-1292,(1989); Capecchi, Trends in Genet. 5:70-76 (1989)).

[0053] Successful recombination may be identified by analyzing the DNAof the selected cells to confirm homologous recombination. Varioustechniques known in the art, such as PCR and/or Southern analysis may beused to confirm homologous recombination events.

[0054] Homologous recombination may also be used to disrupt genes instem cells, and other cell types, which are not totipotent embryonicstem cells. By way of example, stem cells may be myeloid, lymphoid, orneural progenitor and precursor cells. Such transgenic cells may beparticularly useful in the study of DEZ receptor gene function inindividual developmental pathways. Stem cells may be derived from anyvertebrate species, such as mouse, rat, dog, cat, pig, rabbit, human,non-human primates and the like.

[0055] In cells that are not totipotent it may be desirable to knock outboth copies of the target using methods that are known in the art. Forexample, cells comprising homologous recombination at a target locusthat have been selected for expression of a positive selection marker(e.g., Neo^(r)) and screened for non-random integration, can be furtherselected for multiple copies of the selectable marker gene by exposureto elevated levels of the selective agent (e.g., G418). The cells arethen analyzed for homozygosity at the target locus. Alternatively, asecond construct can be generated with a different positive selectionmarker inserted between the two homologous sequences. The two constructscan be introduced into the cell either sequentially or simultaneously,followed by appropriate selection for each of the positive marker genes.The final cell is screened for homologous recombination of both allelesof the target.

[0056] Production of Transgenic Animals

[0057] Selected cells are then injected into a blastocyst (or otherstage of development suitable for the purposes of creating a viableanimal, such as, for example, a morula) of an animal (e.g., a mouse) toform chimeras (see e.g., Bradley, A. in Teratocarcinomas and EmbryonicStem Cells: A Practical Approach, E. J. Robertson, ed., IRL, Oxford, pp.113-152 (1987)). Alternatively, selected ES cells can be allowed toaggregate with dissociated mouse embryo cells to form the aggregationchimera. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term.Chimeric progeny harbouring the homologously recombined DNA in theirgerm cells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA. In one embodiment, chimericprogeny mice are used to generate a mouse with a heterozygous disruptionin the DEZ receptor gene. Heterozygous transgenic mice can then bemated. It is well know in the art that typically ¼ of the offspring ofsuch matings will have a homozygous disruption in the DEZ receptor gene.

[0058] The heterozygous and homozygous transgenic mice can then becompared to normal, wild type mice to determine whether disruption ofthe DEZ receptor gene causes phenotypic changes, especially pathologicalchanges. For example, heterozygous and homozygous mice may be evaluatedfor phenotypic changes by physical examination, necropsy, histology,clinical chemistry, complete blood count, body weight, organ weights,and cytological evaluation of bone marrow.

[0059] In one embodiment, the phenotype (or phenotypic change)associated with a disruption in the DEZ receptor gene is placed into orstored in a database. Preferably, the database includes: (i) genotypicdata (e.g., identification of the disrupted gene) and (ii) phenotypicdata (e.g., phenotype(s) resulting from the gene disruption) associatedwith the genotypic data. The database is preferably electronic. Inaddition, the database is preferably combined with a search tool so thatthe database is searchable.

[0060] Conditional Transgenic Animals

[0061] The present invention further contemplates conditional transgenicor knockout animals, such as those produced using recombination methods.Bacteriophage P1 Cre recombinase and flp recombinase from yeast plasmidsare two non-limiting examples of site-specific DNA recombinase enzymesthat cleave DNA at specific target sites (lox P sites for crerecombinase and frt sites for flp recombinase) and catalyze a ligationof this DNA to a second cleaved site. A large number of suitablealternative site-specific recombinases have been described, and theirgenes can be used in accordance with the method of the presentinvention. Such recombinases include the Int recombinase ofbacteriophage λ (with or without Xis) (Weisberg, R. et al., in LambdaII, (Hendrix, R., et al., Eds.), Cold Spring Harbor Press, Cold SpringHarbor, N.Y., pp. 211-50 (1983), herein incorporated by reference); TpnIand the β-lactamase transposons (Mercier, et al., J. Bacteriol.,172:3745-57 (1990)); the Tn3 resolvase (Flanagan & Fennewald J. Molec.Biol., 206:295-304 (1989); Stark, et al., Cell, 58:779-90 (1989)); theyeast recombinases (Matsuzaki, et al., J. Bacteriol., 172:610-18(1990)); the B. subtilis SpoIVC recombinase (Sato, et al., J. Bacteriol.172:1092-98 (1990)); the Flp recombinase (Schwartz & Sadowski, J.Molec.Biol., 205:647-658 (1989); Parsons, et al., J. Biol. Chem.,265:4527-33 (1990); Golic & Lindquist, Cell, 59:499-509 (1989); Amin, etal., J. Molec. Biol., 214:55-72 (1990)); the Hin recombinase (Glasgow,et al., J. Biol. Chem., 264:10072-82 (1989)); immunoglobulinrecombinases (Malynn, et al., Cell, 54:453-460 (1988)); and the Cinrecombinase (Haffter & Bickle, EMBO J., 7:3991-3996 (1988); Hubner, etal., J. Molec. Biol., 205:493-500 (1989)), all herein incorporated byreference. Such systems are discussed by Echols (J. Biol. Chem.265:14697-14700 (1990)); de Villartay (Nature, 335:170-74 (1988));Craig, (Ann. Rev. Genet., 22:77-105 (1988)); Poyart-Salmeron, et al.,(EMBO J. 8:2425-33 (1989)); Hunger-Bertling, et al., (Mol Cell.Biochem., 92:107-16 (1990)); and Cregg & Madden (Mol. Gen. Genet.,219:320-23 (1989)), all herein incorporated by reference.

[0062] Cre has been purified to homogeneity, and its reaction with theloxP site has been extensively characterized (Abremski & Hess J. Mol.Biol. 259:1509-14 (1984), herein incorporated by reference). Cre proteinhas a molecular weight of 35,000 and can be obtained commercially fromNew England Nuclear/Du Pont. The cre gene (which encodes the Creprotein) has been cloned and expressed (Abremski, et al., Cell32:1301-11 (1983), herein incorporated by reference). The Cre proteinmediates recombination between two loxP sequences (Sternberg, et al.,Cold Spring Harbor Symp. Quant. Biol. 45:297-309 (1981)), which may bepresent on the same or different DNA molecule. Because the internalspacer sequence of the loxP site is asymmetrical, two loxP sites canexhibit directionality relative to one another (Hoess & Abremski Proc.Natl. Acad. Sci. U.S.A. 81:1026-29 (1984)). Thus, when two sites on thesame DNA molecule are in a directly repeated orientation, Cre willexcise the DNA between the sites (Abremski, et al., Cell 32:1301-11(1983)). However, if the sites are inverted with respect to each other,the DNA between them is not excised after recombination but is simplyinverted. Thus, a circular DNA molecule having two loxP sites in directorientation will recombine to produce two smaller circles, whereascircular molecules having two loxP sites in an inverted orientationsimply invert the DNA sequences flanked by the loxP sites. In addition,recombinase action can result in reciprocal exchange of regions distalto the target site when targets are present on separate DNA molecules.

[0063] Recombinases have important application for characterizing genefunction in knockout models. When the constructs described herein areused to disrupt DEZ receptor genes, a fusion transcript can be producedwhen insertion of the positive selection marker occurs downstream (3′)of the translation initiation site of the DEZ receptor gene. The fusiontranscript could result in some level of protein expression with unknownconsequence. It has been suggested that insertion of a positiveselection marker gene can affect the expression of nearby genes. Theseeffects may make it difficult to determine gene function after aknockout event since one could not discern whether a given phenotype isassociated with the inactivation of a gene, or the transcription ofnearby genes. Both potential problems are solved by exploitingrecombinase activity. When the positive selection marker is flanked byrecombinase sites in the same orientation, the addition of thecorresponding recombinase will result in the removal of the positiveselection marker. In this way, effects caused by the positive selectionmarker or expression of fusion transcripts are avoided.

[0064] In one embodiment, purified recombinase enzyme is provided to thecell by direct microinjection. In another embodiment, recombinase isexpressed from a co-transfected construct or vector in which therecombinase gene is operably linked to a functional promoter. Anadditional aspect of this embodiment is the use of tissue-specific orinducible recombinase constructs that allow the choice of when and whererecombination occurs. One method for practicing the inducible forms ofrecombinase-mediated recombination involves the use of vectors that useinducible or tissue-specific promoters or other gene regulatory elementsto express the desired recombinase activity. The inducible expressionelements are preferably operatively positioned to allow the induciblecontrol or activation of expression of the desired recombinase activity.Examples of such inducible promoters or other gene regulatory elementsinclude, but are not limited to, tetracycline, metallothionine,ecdysone, and other steroid-responsive promoters, rapamycin responsivepromoters, and the like (No, et al., Proc. Natl. Acad. Sci. USA,93:3346-51 (1996); Furth, et al., Proc. Natl. Acad. Sci. USA, 91:9302-6(1994)). Additional control elements that can be used include promotersrequiring specific transcription factors such as viral, promoters.Vectors incorporating such promoters would only express recombinaseactivity in cells that express the necessary transcription factors.

[0065] Models for Disease

[0066] The cell- and animal-based systems described herein can beutilized as models for diseases. Animals of any species, including, butnot limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs,goats, and non-human primates, e.g., baboons, monkeys, and chimpanzeesmay be used to generate disease animal models. In addition, cells fromhumans may be used. These systems may be used in a variety ofapplications. Such assays may be utilized as part of screeningstrategies designed to identify agents, such as compounds that arecapable of ameliorating disease symptoms. Thus, the animal- andcell-based models may be used to identify drugs, pharmaceuticals,therapies and interventions that may be effective in treating disease.

[0067] Cell-based systems may be used to identify compounds that may actto ameliorate disease symptoms. For example, such cell systems may beexposed to a compound suspected of exhibiting an ability to amelioratedisease symptoms, at a sufficient concentration and for a timesufficient to elicit such an amelioration of disease symptoms in theexposed cells. After exposure, the cells are examined to determinewhether one or more of the disease cellular phenotypes has been alteredto resemble a more normal or more wild type, non-disease phenotype.

[0068] In addition, animal-based disease systems, such as thosedescribed herein, may be used to identify compounds capable ofameliorating disease symptoms. Such animal models may be used as testsubstrates for the identification of drugs, pharmaceuticals, therapies,and interventions that may be effective in treating a disease or otherphenotypic characteristic of the animal. For example, animal models maybe exposed to a compound or agent suspected of exhibiting an ability toameliorate disease symptoms, at a sufficient concentration and for atime sufficient to elicit such an amelioration of disease symptoms inthe exposed animals. The response of the animals to the exposure may bemonitored by assessing the reversal of disorders associated with thedisease. Exposure may involve treating mother animals during gestationof the model animals described herein, thereby exposing embryos orfetuses to the compound or agent that may prevent or ameliorate thedisease or phenotype. Neonatal, juvenile, and adult animals can also beexposed.

[0069] More particularly, using the animal models of the invention,specifically, transgenic mice, methods of identifying agents, includingcompounds are provided, preferably, on the basis of the ability toaffect at least one phenotype associated with a disruption in a DEZreceptor gene. In one embodiment, the present invention provides amethod of identifying agents having an effect on DEZ receptor expressionor function. The method includes measuring a physiological response ofthe animal, for example, to the agent, and comparing the physiologicalresponse of such animal to a control animal, wherein the physiologicalresponse of the animal comprising a disruption in a DEZ receptor ascompared to the control animal indicates the specificity of the agent. A“physiological response” is any biological or physical parameter of ananimal that can be measured. Molecular assays (e.g., gene transcription,protein production and degradation rates), physical parameters (e.g.,exercise physiology tests, measurement of various parameters ofrespiration, measurement of heart rate or blood pressure, measurement ofbleeding time, aPTT.T, or TT), and cellular assays (e.g.,.immunohistochemical assays of cell surface markers, or the ability ofcells to aggregate or proliferate) can be used to assess a physiologicalresponse. The transgenic animals and cells of the present invention maybe utilized as models for diseases, disorders, or conditions associatedwith phenotypes relating to a disruption in a DEZ receptor.

[0070] The present invention provides a unique animal model for testingand developing new treatments relating to the behavioral phenotypes.Analysis of the behavioral phenotype allows for the development of ananimal model useful for testing, for instance, the efficacy of proposedgenetic and pharmacological therapies for human genetic diseases, suchas neurological, neuropsychological, or psychotic illnesses.

[0071] A statistical analysis of the various behaviors measured can becarried out using any conventional statistical program routinely used bythose skilled in the art (such as, for example, “Analysis of Variance”or ANOVA). A “p” value of about 0.05 or less is generally considered tobe statistically significant, although slightly higher p values maystill be indicative of statistically significant differences. Tostatistically analyze abnormal behavior, a comparison is made betweenthe behavior of a transgenic animal (or a group thereof) to the behaviorof a wild-type mouse (or a group thereof), typically under certainprescribed conditions. “Abnormal behavior” as used herein refers tobehavior exhibited by an animal having a disruption in the DEZ receptorgene, e.g. transgenic animal, which differs from an animal without adisruption in the DEZ receptor gene, e.g. wild-type mouse. Abnormalbehavior consists of any number of standard behaviors that can beobjectively measured (or observed) and compared. In the case ofcomparison, it is preferred that the change be statistically significantto confirm that there is indeed a meaningful behavioral differencebetween the knockout animal and the wild-type control animal. Examplesof behaviors that may be measured or observed include, but are notlimited to, ataxia, rapid limb movement, eye movement, breathing, motoractivity, cognition, emotional behaviors, social behaviors,hyperactivity, hypersensitivity, anxiety, impaired learning, abnormalreward behavior, and abnormal social interaction, such as aggression.

[0072] A series of tests may be used to measure the behavioral phenotypeof the animal models of the present invention, including neurologicaland neuropsychological tests to identify abnormal behavior. These testsmay be used to measure abnormal behavior relating to, for example,learning and memory, eating, pain, aggression, sexual reproduction,anxiety, depression, schizophrenia, and drug abuse. (see, e.g., Crawley& Paylor, Hormones and Behavior 31:197-211 (1997)).

[0073] The social interaction test involves exposing a mouse to otheranimals in a variety of settings. The social behaviors of the animals(e.g., touching, climbing, sniffing, and mating) are subsequentlyevaluated. Differences in behaviors can then be statistically analyzedand compared (see, e.g., S. E. File, et al., Pharmacol. Bioch. Behav.22:941-944 (1985); R. R. Holson, Phys. Behav. 37:239-247 (1986)).Examplary behavioral tests include the following.

[0074] The mouse startle response test typically involves exposing theanimal to a sensory (typically auditory) stimulus and measuring thestartle response of the animal (see, e.g., M. A. Geyer, et al., BrainRes. Bull. 25:485-498 (1990); Paylor and Crawley, Psychopharmacology132:169-180 (1997)). A pre-pulse inhibition test can also be used, inwhich the percent inhibition (from a normal startle response) ismeasured by “cueing” the animal first with a brief low-intensitypre-pulse prior to the startle pulse.

[0075] The electric shock test generally involves exposure to anelectrified surface and measurement of subsequent behaviors such as, forexample, motor activity, learning, social behaviors. The behaviors aremeasured and statistically analyzed using standard statistical tests.(see, e.g., G. J. Kant, et al., Pharm. Bioch. Behav. 20:793-797 (1984);N. J. Leidenheimer, et al., Pharmacol. Bioch. Behav. 30:351-355 (1988)).

[0076] The tail-pinch or immobilization test involves applying pressureto the tail of the animal and/or restraining the animal's movements.Motor activity, social behavior, and cognitive behavior are examples ofthe areas that are measured. (see, e.g., M. Bertolucci D'Angic, et al.,Neurochem. 55:1208-1214 (1990)).

[0077] The novelty test generally comprises exposure to a novelenvironment and/or novel objects. The animal's motor behavior in thenovel environment and/or around the novel object are measured andstatistically analyzed. (see, e.g., D. K. Reinstein, et al., Pharm.Bioch. Behav. 17:193-202 (1982); B. Poucet, Behav. Neurosci.103:1009-10016 (1989); R. R. Holson, et al., Phys. Behav. 37:231-238(1986)). This test may be used to detect visual processing deficienciesor defects.

[0078] The learned helplessness test involves exposure to stresses, forexample, noxious stimuli, which cannot be affected by the animal'sbehavior. The animal's behavior can be statistically analyzed usingvarious standard statistical tests. (see, e.g., A. Leshner, et al.,Behav. Neural Biol. 26:497-501 (1979)).

[0079] Alternatively, a tail suspension test may be used, in which the“immobile” time of the mouse is measured when suspended “upside-down” byits tail. This is a measure of whether the animal struggles, anindicator of depression. In humans, depression is believed to resultfrom feelings of a lack of control over one's life or situation. It isbelieved that a depressive state can be elicited in animals byrepeatedly subjecting them to aversive situations over which they haveno control. A condition of “learned helplessness” is eventually reached,in which the animal will stop trying to change its circumstances andsimply accept its fate. Animals that stop struggling sooner are believedto be more prone to depression. Studies have shown that theadministration of certain antidepressant drugs prior to testingincreases the amount of time that animals struggle before giving up.

[0080] The Morris water-maze test comprises learning spatialorientations in water and subsequently measuring the animal's behaviors,such as, for example, by counting the number of incorrect choices. Thebehaviors measured are statistically analyzed using standard statisticaltests. (see, e.g., E. M. Spruijt, et al., Brain Res. 527:192-197(1990)).

[0081] Alternatively, a Y-shaped maze may be used (see, e.g., McFarland,D. J., Pharmacology, Biochemistry and Behavior 32:723-726 (1989); Dellu,F., et al., Neurobiology of Learning and Memory 73:31-48 (2000)). TheY-maze is generally believed to be a test of cognitive ability. Thedimensions of each arm of the Y-maze can be, for example, approximately40 cm×8 cm×20 cm, although other dimensions may be used. Each arm canalso have, for example, sixteen equally spaced photobeams toautomatically detect movement within the arms. At least two differenttests can be performed using such a Y-maze. In a continuous Y-mazeparadigm, mice are allowed to explore all three arms of a Y-maze for,e.g., approximately 10 minutes. The animals are continuously trackedusing photobeam detection grids, and the data can be used to measurespontaneous alteration and positive bias behavior. Spontaneousalteration refers to the natural tendency of a “normal” animal to visitthe least familiar arm of a maze. An alternation is scored when theanimal makes two consecutive turns in the same direction, thusrepresenting a sequence of visits to the least recently entered arm ofthe maze. Position bias determines egocentrically defined responses bymeasuring the animal's tendency to favor turning in one direction overanother. Therefore, the test can detect differences in an animal'sability to navigate on the basis of allocentric or egocentricmechanisms. The two-trial Y-maze memory test measures response tonovelty and spatial memory based on a free-choice exploration paradigm.During the first trial (acquisition), the animals are allowed to freelyvisit two arms of the Y-maze for, e.g., approximately 15 minutes. Thethird arm is blocked off during this trial. The second trial (retrieval)is performed after an intertrial interval of, e.g., approximately 2hours. During the retrieval trial, the blocked arm is opened and theanimal is allowed access to all three arms for, e.g., approximately 5minutes. Data are collected during the retrieval trial and analyzed forthe number and duration of visits to each arm. Because the three arms ofthe maze are virtually identical, discrimination between novelty andfamiliarity is dependent on “environmental” spatial cues around the roomrelative to the position of each arm. Changes in arm entry and durationof time spent in the novel arm in a transgenic animal model may beindicative of a role of that gene in mediating novelty and recognitionprocesses.

[0082] The passive avoidance or shuttle box test generally involvesexposure to two or more environments, one of which is noxious, providinga choice to be learned by the animal. Behavioral measures include, forexample, response latency, number of correct responses, and consistencyof response. (see, e.g., R. Ader, et al., Psychon. Sci. 26:125-128(1972); R. R. Holson, Phys. Behav. 37:221-230 (1986)). Alternatively, azero maze can be used. In a zero-maze, the animals can, for example, beplaced in a closed quadrant of an elevated annular platform having,e.g., 2 open and 2 closed quadrants, and are allowed to explore forapproximately 5 minutes. This paradigm exploits an approach-avoidanceconflict between normal exploratory activity and an aversion to openspaces in rodents. This test measures anxiety levels and can be used toevaluate the effectiveness of anti-anxiolytic drugs. The time spent inopen quadrants versus closed quadrants may be recorded automatically,with, for example, the placement of photobeams at each transition site.

[0083] The food avoidance test involves exposure to novel food andobjectively measuring, for example, food intake and intake latency. Thebehaviors measured are statistically analyzed using standard statisticaltests. (see, e.g., B. A. Campbell, et al., J. Comp. Physiol. Psychol.67:15-22 (1969)).

[0084] The elevated plus-maze test comprises exposure to a maze, withoutsides, on a platform, the animal's behavior is objectively measured bycounting the number of maze entries and maze learning. The behavior isstatistically analyzed using standard statistical tests. (see, e.g., H.A. Baldwin, et al., Brain Res. Bull, 20:603-606 (1988)).

[0085] The stimulant-induced hyperactivity test involves injection ofstimulant drugs (e.g., amphetamines, cocaine, PCP, and the like), andobjectively measuring, for example, motor activity, social interactions,cognitive behavior. The animal's behaviors are statistically analyzedusing standard statistical tests. (see, e.g., P. B. S. Clarke, et al.,Psychopharmacology 96:511-520 (1988); P. Kuczenski, etal., J.Neuroscience 11:2703-2712 (1991)).

[0086] The self-stimulation test generally comprises providing the mousewith the opportunity to regulate electrical and/or chemical stimuli toits own brain. Behavior is measured by frequency and pattern ofself-stimulation. Such behaviors are statistically analyzed usingstandard statistical tests. (see, e.g., S. Nassif, et al., Brain Res.,332:247-257 (1985); W. L. Isaac, et al., Behav. Neurosci. 103:345-355(1989)).

[0087] The reward test involves shaping a variety of behaviors, e.g.,motor, cognitive, and social, measuring, for example, rapidity andreliability of behavioral change, and statistically analyzing thebehaviors measured. (see, e.g., L. E. Jarrard, et al., Exp. Brain Res.61:519-530 (1986)).

[0088] The DRL (differential reinforcement to low rates of responding)performance test involves exposure to intermittent reward paradigms andmeasuring the number of proper responses, e.g., lever pressing. Suchbehavior is statistically analyzed using standard statistical tests.(see, e.g., J. D. Sinden, et al., Behav. Neurosci. 100:320-329 (1986);V. Nalwa, et al., Behav Brain Res. 17:73-76 (1985); and A. J. Nonneman,et al., J. Comp. Physiol. Psych. 95:588-602 (1981)).

[0089] The spatial learning test involves exposure to a complex novelenvironment, measuring the rapidity and extent of spatial learning, andstatistically analyzing the behaviors measured. (see, e.g., N. Pitsikas,et al., Pharm. Bioch. Behav. 38:931-934 (1991); B. Poucet, et al., BrainRes. 37:269-280 (1990); D. Christie, et al., Brain Res. 37:263-268(1990); and F. Van Haaren, et al., Behav. Neurosci. 102:481-488 (1988)).Alternatively, an open-field (of) test may be used, in which the greaterdistance traveled for a given amount of time is a measure of theactivity level and anxiety of the animal. When the open field is a novelenvironment, it is believed that an approach-avoidance situation iscreated, in which the animal is “torn” between the drive to explore andthe drive to protect itself. Because the chamber is lighted and has noplaces to hide other than the comers, it is expected that a “normal”mouse will spend more time in the comers and around the periphery thanit will in the center where there is no place to hide. “Normal” micewill, however, venture into the central regions as they explore more andmore of the chamber. It can then be extrapolated that especially anxiousmice will spend most of their time in the corners, with relativelylittle or no exploration of the central region, whereas bold (i.e., lessanxious) mice will travel a greater distance, showing little preferencefor the periphery versus the central region.

[0090] The visual, somatosensory and auditory neglect tests generallycomprise exposure to a sensory stimulus, objectively measuring, forexample, orientating responses, and statistically analyzing thebehaviors measured. (see, e.g., J. M. Vargo, et al., Exp. Neurol.102:199-209 (1988)).

[0091] The consummatory behavior test generally comprises feeding anddrinking, and objectively measuring quantity of consumption. Thebehavior measured is statistically analyzed using standard statisticaltests. (see, e.g., P. J. Fletcher, et al., Psychopharmacol. 102:301-308(1990); M. G. Corda, et al., Proc. Nat'l Acad. Sci. USA 80:2072-2076(1983)).

[0092] A visual discrimination test can also be used to evaluate thevisual processing of an animal. One or two similar objects are placed inan open field and the animal is allowed to explore for about 5-10minutes. The time spent exploring each object (proximity to, i.e.,movement within, e.g., about 3-5 cm of the object is consideredexploration of an object) is recorded. The animal is then removed fromthe open field, and the objects are replaced by a similar object and anovel object. The animal is returned to the open field and the percenttime spent exploring the novel object over the old object is measured(again, over about a 5-10 minute span). “Normal” animals will typicallyspend a higher percentage of time exploring the novel object rather thanthe old object. If a delay is imposed between sampling and testing, thememory task becomes more hippocampal-dependent. If no delay is imposed,the task is more based on simple visual discrimination. This test canalso be used for olfactory discrimination, in which the objects(preferably, simple blocks) can be sprayed or otherwise treated to holdan odor. This test can also be used to determine if the animal can makegustatory discriminations; animals that return to the previously eatenfood instead of novel food exhibit gustatory neophobia.

[0093] A hot plate analgesia test can be used to evaluate an animal'ssensitivity to heat or painful stimuli. For example, a mouse can beplaced on an approximately 55° C. hot plate and the mouse's responselatency (e.g., time to pick up and lick a hind paw) can be recorded.These responses are not reflexes, but rather “higher” responsesrequiring cortical involvement. This test may be used to evaluate anociceptive disorder.

[0094] An accelerating rotarod test may be used to measure coordinationand balance in mice. Animals can be, for example, placed on a rod thatacts like a rotating treadmill (or rolling log). The rotarod can be madeto rotate slowly at first and then progressively faster until it reachesa speed of, e.g., approximately 60 rpm. The mice must continuallyreposition themselves in order to avoid falling off. The animals arepreferably tested in at least three trials, a minimum of 20 minutesapart. Those mice that are able to stay on the rod the longest arebelieved to have better coordination and balance.

[0095] A metrazol administration test can be used to screen animals forvarying susceptibilities to seizures or similar events. For example, a 5mg/ml solution of metrazol can be infused through the tail vein of amouse at a rate of, e.g., approximately 0.375 ml/min. The infusion willcause all mice to experience seizures, followed by death. Those micethat enter the seizure stage the soonest are believed to be more proneto seizures. Four distinct physiological stages can be recorded: soonafter the start of infusion, the mice will exhibit a noticeable“twitch”, followed by a series of seizures, ending in a final tensing ofthe body known as “tonic extension”, which is followed by death.

[0096] DEZ Receptor Gene Products

[0097] The present invention further contemplates use of the DEZreceptor gene sequence to produce DEZ receptor gene products. DEZreceptor gene products may include proteins that represent functionallyequivalent gene products. Such an equivalent gene product may containdeletions, additions or substitutions of amino acid residues within theamino acid sequence encoded by the gene sequences described herein, butwhich result in a silent change, thus producing a functionallyequivalent DEZ receptor gene product. Amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved.

[0098] For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. “Functionally equivalent”, as utilized herein, refers toa protein capable of exhibiting a substantially similar in vivo activityas the endogenous gene products encoded by the DEZ receptor genesequences. Alternatively, when utilized as part of an assay,“functionally equivalent” may refer to peptides capable of interactingwith other cellular or extracellular molecules in a manner substantiallysimilar to the way in which the corresponding portion of the endogenousgene product would.

[0099] Other protein products useful according to the methods of theinvention are peptides derived from or based on the DEZ receptor geneproduced by recombinant or synthetic means (derived peptides).

[0100] DEZ receptor gene products may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing the gene polypeptides and peptides of the invention byexpressing nucleic acid encoding gene sequences are described herein.Methods that are well known to those skilled in the art can be used toconstruct expression vectors containing gene protein coding sequencesand appropriate transcriptional/translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques and in vivo recombination/genetic recombination(see, e.g., Sambrook, et al., 1989, supra, and Ausubel, et al., 1989,supra). Alternatively, RNA capable of encoding gene protein sequencesmay be chemically synthesized using, for example, automated synthesizers(see, e.g. Oligonucleotide Synthesis: A Practical Approach, Gait, M. J.ed., IRL Press, Oxford (1984)).

[0101] A variety of host-expression vector systems may be utilized toexpress the gene coding sequences of the invention. Such host-expressionsystems represent vehicles by which the coding sequences of interest maybe produced and subsequently purified, but also represent cells thatmay, when transformed or transfected with the appropriate nucleotidecoding sequences, exhibit the gene protein of the invention in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing geneprotein coding sequences; yeast (e.g. Saccharomyces, Pichia) transformedwith recombinant yeast expression vectors containing the gene proteincoding sequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the gene proteincoding sequences; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors(e.g., Ti plasmid) containing gene protein coding sequences; ormammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionine promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5 K promoter).

[0102] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the geneprotein being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of antibodies or to screenpeptide libraries, for example, vectors that direct the expression ofhigh levels of fusion protein products that are readily purified may bedesirable. Such vectors include, but are not limited, to the E. coliexpression vector pUR278 (Ruther et al., EMBO J., 2:1791-94 (1983)), inwhich the gene protein coding sequence may be ligated individually intothe vector in frame with the lac Z coding region so that a fusionprotein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.,13:3101-09 (1985); Van Heeke et al., J. Biol. Chem., 264:5503-9 (1989));and the like. pGEX vectors may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned DEZ receptor gene protein can be released from the GSTmoiety.

[0103] In a preferred embodiment, full length cDNA sequences areappended with in-frame Bam HI sites at the amino terminus and Eco RIsites at the carboxyl terminus using standard PCR methodologies (Innis,et al. (eds) PCR Protocols: A Guide to Methods and Applications,Academic Press, San Diego (1990)) and ligated into the pGEX-2TK vector(Pharmacia, Uppsala, Sweden). The resulting cDNA construct contains akinase recognition site at the amino terminus for radioactive labelingand glutathione S-transferase sequences at the carboxyl terminus foraffinity purification (Nilsson, et al., EMBO J., 4: 1075-80 (1985);Zabeau et al., EMBO J., 1: 1217-24 (1982)).

[0104] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The gene coding sequence may becloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter). Successful insertion ofgene coding sequence will result in inactivation of the polyhedrin geneand production of non-occluded recombinant virus (i.e., virus lackingthe proteinaceous coat coded for by the polyhedrin gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted gene is expressed (see, e.g., Smith, et al., J.Virol. 46: 584-93 (1983); U.S. Pat. No. 4,745,051).

[0105] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the gene coding sequence of interest may be ligatedto an adenovirus transcription/translation control complex, e.g., thelate promoter and tripartite leader sequence. This chimeric gene maythen be inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing gene protein in infected hosts. (e.g.,see Logan et al., Proc. Natl. Acad. Sci. USA, 81:3655-59 (1984)).Specific initiation signals may also be required for efficienttranslation of inserted gene coding sequences. These signals include theATG initiation codon and adjacent sequences. In cases where an entiregene, including its own initiation codon and adjacent sequences, isinserted into the appropriate expression vector, no additionaltranslational control signals may be needed. However, in cases whereonly a portion of the gene coding sequence is inserted, exogenoustranslational control signals, including, perhaps, the ATG initiationcodon, must be provided. Furthermore, the initiation codon must be inphase with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bitter, et al., Methods inEnzymol., 153:516-44 (1987)).

[0106] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellsthat possess the cellular machinery for proper processing of the primarytranscript, glycosylation, and phosphorylation of the gene product maybe used. Such mammalian host cells include but are not limited to CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, W138, etc.

[0107] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines that stablyexpress the gene protein may be engineered. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells that stablyintegrate the plasmid into their chromosomes and grow, to form foci,which in turn can be cloned and expanded into cell lines. This methodmay advantageously be used to engineer cell lines that express the geneprotein. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the gene protein.

[0108] In a preferred embodiment, timing and/or quantity of expressionof the recombinant protein can be controlled using an inducibleexpression construct. Inducible constructs and systems for inducibleexpression of recombinant proteins will be well known to those skilledin the art. Examples of such inducible promoters or other generegulatory elements include, but are not limited to, tetracycline,metallothionine, ecdysone, and other steroid-responsive promoters,rapamycin responsive promoters, and the like (No, et al., Proc. Natl.Acad. Sci. USA, 93:3346-51 (1996); Furth, et al., Proc. Natl. Acad. Sci.USA, 91:9302-6 (1994)). Additional control elements that can be usedinclude promoters requiring specific transcription factors such asviral, particularly HIV, promoters. In one in embodiment, a Tetinducible gene expression system is utilized. (Gossen et al., Proc.Natl. Acad. Sci. USA, 89:5547-51 (1992); Gossen, et al., Science,268:1766-69 (1995)). Tet Expression Systems are based on two regulatoryelements derived from the tetracycline-resistance operon of the E. coliTn10 transposon—the tetracycline repressor protein (TetR) and thetetracycline operator sequence (tetO) to which TetR binds. Using such asystem, expression of the recombinant protein is placed under thecontrol of the tetO operator sequence and transfected or transformedinto a host cell. In the presence of TetR, which is co-transfected intothe host cell, expression of the recombinant protein is repressed due tobinding of the TetR protein to the tetO regulatory element. High-level,regulated gene expression can then be induced in response to varyingconcentrations of tetracycline (Tc) or Tc derivatives such asdoxycycline (Dox), which compete with tetO elements for binding to TetR.Constructs and materials for tet inducible gene expression are availablecommercially from CLONTECH Laboratories, Inc., Palo Alto, Calif.

[0109] When used as a component in an assay system, the gene protein maybe labeled, either directly or indirectly, to facilitate detection of acomplex formed between the gene protein and a test substance. Any of avariety of suitable labeling systems may be used including but notlimited to radioisotopes such as ¹²⁵I; enzyme labeling systems thatgenerate a detectable calorimetric signal or light when exposed tosubstrate; and fluorescent labels. Where recombinant DNA technology isused to produce the gene protein for such assay systems, it may beadvantageous to engineer fusion proteins that can facilitate labeling,immobilization and/or detection.

[0110] Indirect labeling involves the use of a protein, such as alabeled antibody, which specifically binds to the gene product. Suchantibodies include but are not limited to polyclonal, monoclonal,chimeric, single chain, Fab fragments and fragments produced by a Fabexpression library.

[0111] Production of Antibodies

[0112] Described herein are methods for the production of antibodiescapable of specifically recognizing one or more epitopes. Suchantibodies may include, but are not limited to polyclonal antibodies,monoclonal antibodies (mAbs), humanized or chimeric antibodies, singlechain antibodies, Fab fragments, F(ab′)₂ fragments, fragments producedby a Fab expression library, anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above. Such antibodies may beused, for example, in the detection of a DEZ receptor gene in abiological sample, or, alternatively, as a method for the inhibition ofabnormal DEZ receptor gene activity. Thus, such antibodies may beutilized as part of disease treatment methods, and/or may be used aspart of diagnostic techniques whereby patients may be tested forabnormal levels of DEZ receptor gene proteins, or for the presence ofabnormal forms of such proteins.

[0113] For the production of antibodies, various host animals may beimmunized by injection with the DEZ receptor gene, its expressionproduct or a portion thereof. Such host animals may include but are notlimited to rabbits, mice, rats, goats and chickens, to name but a few.Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum.

[0114] Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as DEZ receptor gene product, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, host animals suchas those described above, may be immunized by injection with geneproduct supplemented with adjuvants as also described above.

[0115] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, may be obtained by any techniquethat provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to thehybridoma technique of Kohler and Milstein, Nature, 256:495-7 (1975);and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor, et al., Immunology Today, 4:72 (1983); Cote, et al., Proc.Natl. Acad. Sci. USA, 80:2026-30 (1983)), and the EBV-hybridomatechnique (Cole, et al., in Monoclonal Antibodies And Cancer Therapy,Alan R. Liss, Inc., New York, pp. 77-96 (1985)). Such antibodies may beof any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and anysubclass thereof. The hybridoma producing the mAb of this invention maybe cultivated in vitro or in vivo. Production of high titers of mAbs invivo makes this the presently preferred method of production.

[0116] In addition, techniques developed for the production of “chimericantibodies” (Morrison, et al., Proc. Natl. Acad. Sci., 81:6851-6855(1984); Takeda, et al., Nature, 314:452-54 (1985)) by splicing the genesfrom a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity can be used. A chimeric antibody is a molecule inwhich different portions are derived from different animal species, suchas those having a variable region derived from a murine mAb and a humanimmunoglobulin constant region.

[0117] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-26(1988); Huston, et al., Proc. Natl. Acad. Sci. USA, 85:5879-83 (1988);and Ward, et al., Nature, 334:544-46 (1989)) can be adapted to producegene-single chain antibodies. Single chain antibodies are typicallyformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge, resulting in a single chain polypeptide.

[0118] Antibody fragments that recognize specific epitopes may begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule and the Fab fragments that can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse, etal., Science, 246:1275-81 (1989)) to allow rapid and easy identificationof monoclonal Fab fragments with the desired specificity.

[0119] Screening Methods

[0120] The present invention may be employed in a process for screeningfor agents such as agonists, i.e. agents that bind to and activate DEZreceptor polypeptides, or antagonists, i.e. inhibit the activity orinteraction of DEZ receptor polypeptides with its ligand. Thus,polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures as knownin the art. Any methods routinely used to identify and screen for agentsthat can modulate receptors may be used in accordance with the presentinvention.

[0121] The present invention provides methods for identifying andscreening for agents that modulate DEZ receptor expression or function.More particularly, cells that contain and express DEZ receptor genesequences may be used to screen for therapeutic agents. Such cells mayinclude non-recombinant monocyte cell lines, such as U937 (ATCC#CRL-1593), THP-1 (ATCC# TIB-202), and P388D1 (ATCC# TIB-63); endothelialcells such as HUVEC's and bovine aortic endothelial cells (BAEC's); aswell as generic mammalian cell lines such as HeLa cells and COS cells,e.g., COS-7 (ATCC# CRL-1651). Further, such cells may includerecombinant, transgenic cell lines. For example, the transgenic mice ofthe invention may be used to generate cell lines, containing one or morecell types involved in a disease, that can be used as cell culturemodels for that disorder. While cells, tissues, and primary culturesderived from the disease transgenic animals of the invention may beutilized, the generation of continuous cell lines is preferred. Forexamples of techniques that may be used to derive a continuous cell linefrom the transgenic animals, see Small, et al., Mol. Cell Biol.,5:642-48 (1985).

[0122] DEZ receptor gene sequences may be introduced into, andoverexpressed in, the genome of the cell of interest. In order tooverexpress a DEZ receptor gene sequence, the coding portion of the DEZreceptor gene sequence may be ligated to a regulatory sequence that iscapable of driving gene expression in the cell type of interest. Suchregulatory regions will be well known to those of skill in the art, andmay be utilized in the absence of undue experimentation. DEZ receptorgene sequences may also be disrupted or underexpressed. Cells having DEZreceptor gene disruptions or underexpressed DEZ receptor gene sequencesmay be used, for example, to screen for agents capable of affectingalternative pathways that compensate for any loss of functionattributable to the disruption or underexpression.

[0123] In vitro systems may be designed to identify compounds capable ofbinding the DEZ receptor gene products. Such compounds may include, butare not limited to, peptides made of D- and/or L-configuration aminoacids (in, for example, the form of random peptide libraries; (see e.g.,Lam, et al., Nature, 354:82-4 (1991)), phosphopeptides (in, for example,the form of random or partially degenerate, directed phosphopeptidelibraries; see, e.g., Songyang, et al., Cell, 72:767-78 (1993)),antibodies, and small organic or inorganic molecules. Compoundsidentified may be useful, for example, in modulating the activity of DEZreceptor gene proteins, preferably mutant DEZ receptor gene proteins;elaborating the biological function of the DEZ receptor gene protein; orscreening for compounds that disrupt normal DEZ receptor geneinteractions or themselves disrupt such interactions.

[0124] The principle of the assays used to identify compounds that bindto the DEZ receptor gene protein involves preparing a reaction mixtureof the DEZ receptor gene protein and the test compound under conditionsand for a time sufficient to allow the two components to interact andbind, thus forming a complex that can be removed and/or detected in thereaction mixture. These assays can be conducted in a variety of ways.For example, one method to conduct such an assay would involve anchoringthe DEZ receptor gene protein or the test substance onto a solid phaseand detecting target protein/test substance complexes anchored on thesolid phase at the end of the reaction. In one embodiment of such amethod, the DEZ receptor gene protein may be anchored onto a solidsurface, and the test compound, which is not anchored, may be labeled,either directly or indirectly.

[0125] In practice, microtitre plates are conveniently utilized. Theanchored component may be immobilized by non-covalent or covalentattachments. Non-covalent attachment may be accomplished simply bycoating the solid surface with a solution of the protein and drying.Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein may be used to anchor the protein tothe solid surface. The surfaces may be prepared in advance and stored.

[0126] In order to conduct the assay, the nonimmobilized component isadded to the coated surface containing the anchored component. After thereaction is complete, unreacted components are removed (e.g., bywashing) under conditions such that any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thepreviously nonimmobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the previously nonimmobilized component is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the previouslynonimmobilized component (the antibody, in turn, may be directly labeledor indirectly labeled with a labeled anti-Ig antibody).

[0127] Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for DEZ receptorgene product or the test compound to anchor any complexes formed insolution, and a labeled antibody specific for the other component of thepossible complex to detect anchored complexes.

[0128] Compounds that are shown to bind to a particular DEZ receptorgene product through one of the methods described above can be furthertested for their ability to elicit a biochemical response from the DEZreceptor gene protein. Agonists, antagonists and/or inhibitors of theexpression product can be identified utilizing assays well known in theart.

[0129] Antisense, Ribozymes, and Antibodies

[0130] Other agents that may be used as therapeutics include the DEZreceptor gene, its expression product(s) and functional fragmentsthereof. Additionally, agents that reduce or inhibit mutant DEZ receptorgene activity may be used to ameliorate disease symptoms. Such agentsinclude antisense, ribozyme, and triple helix molecules. Techniques forthe production and use of such molecules are well known to those ofskill in the art.

[0131] Anti-sense RNA and DNA molecules act to directly block thetranslation of mRNA by hybridizing to targeted mRNA and preventingprotein translation. With respect to antisense DNA,oligodeoxyribonucleotides derived from the translation initiation site,e.g., between the −10 and +10 regions of the DEZ receptor genenucleotide sequence of interest, are preferred.

[0132] Ribozymes are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by an endonucleolytic cleavage. Thecomposition of ribozyme molecules must include one or more sequencescomplementary to the DEZ receptor gene mRNA, and must include the wellknown catalytic sequence responsible for mRNA cleavage. For thissequence, see U.S. Pat. No. 5,093,246, which is incorporated byreference herein in its entirety. As such within the scope of theinvention are engineered hammerhead motif ribozyme molecules thatspecifically and efficiently catalyze endonucleolytic cleavage of RNAsequences encoding DEZ receptor gene proteins.

[0133] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the molecule of interest forribozyme cleavage sites that include the following sequences, GUA, GUUand GUC. Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the DEZ receptor genecontaining the cleavage site may be evaluated for predicted structuralfeatures, such as secondary structure, that may render theoligonucleotide sequence unsuitable. The suitability of candidatesequences may also be evaluated by testing their accessibility tohybridization with complementary oligonucleotides, using ribonucleaseprotection assays.

[0134] Nucleic acid molecules to be used in triple helix formation forthe inhibition of transcription should be single stranded and composedof deoxyribonucleotides. The base composition of these oligonucleotidesmust be designed to promote triple helix formation via Hoogsteen basepairing rules, which generally require sizeable stretches of eitherpurines or pyrimidines to be present on one strand of a duplex.Nucleotide sequences may be pyrimidine-based, which will result in TATand CGC triplets across the three associated strands of the resultingtriple helix. The pyrimidine-rich molecules provide base complementarityto a purine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in GGCtriplets across the three strands in the triplex.

[0135] Alternatively, the potential sequences that can be targeted fortriple helix formation may be increased by creating a so called“switchback” nucleic acid molecule. Switchback molecules are synthesizedin an alternating 5′-3′, 3′-5′ manner, such that they base pair withfirst one strand of a duplex and then the other, eliminating thenecessity for a sizeable stretch of either purines or pyrimidines to bepresent on one strand of a duplex.

[0136] It is possible that the antisense, ribozyme, and/or triple helixmolecules described herein may reduce or inhibit the transcription(triple helix) and/or translation (antisense, ribozyme) of mRNA producedby both normal and mutant DEZ receptor gene alleles. In order to ensurethat substantially normal levels of DEZ receptor gene activity aremaintained, nucleic acid molecules that encode and express DEZ receptorgene polypeptides exhibiting normal activity may be introduced intocells that do not contain sequences susceptible to whatever antisense,ribozyme, or triple helix treatments are being utilized. Alternatively,it may be preferable to coadminister normal DEZ receptor gene proteininto the cell or tissue in order to maintain the requisite level ofcellular or tissue DEZ receptor gene activity.

[0137] Anti-sense RNA and DNA, ribozyme, and triple helix molecules ofthe invention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

[0138] Various well-known modifications to the DNA molecules may beintroduced as a means of increasing intracellular stability andhalf-life. Possible modifications include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule or the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages within the oligodeoxyribonucleotide backbone.

[0139] Antibodies that are both specific for DEZ receptor gene protein,and in particular, mutant gene protein, and interfere with its activitymay be used to inhibit mutant DEZ receptor gene function. Suchantibodies may be generated against the proteins themselves or againstpeptides corresponding to portions of the proteins using standardtechniques known in the art and as also described herein. Suchantibodies include but are not limited to polyclonal, monoclonal, Fabfragments, single chain antibodies, chimeric antibodies, etc.

[0140] In instances where the DEZ receptor gene protein is intracellularand whole antibodies are used, internalizing antibodies may bepreferred. However, lipofectin liposomes may be used to deliver theantibody or a fragment of the Fab region that binds to the DEZ receptorgene epitope into cells. Where fragments of the antibody are used, thesmallest inhibitory fragment that binds to the target or expanded targetprotein's binding domain is preferred. For example, peptides having anamino acid sequence corresponding to the domain of the variable regionof the antibody that binds to the DEZ receptor gene protein may be used.Such peptides may be synthesized chemically or produced via recombinantDNA technology using methods well known in the art (see, e.g.,Creighton, Proteins: Structures and Molecular Principles (1984) W. H.Freeman, New York 1983, supra; and Sambrook, et al., 1989, supra).Alternatively, single chain neutralizing antibodies that bind tointracellular DEZ receptor gene epitopes may also be administered. Suchsingle chain antibodies may be administered, for example, by expressingnucleotide sequences encoding single-chain antibodies within the targetcell population by utilizing, for example, techniques such as thosedescribed in Marasco, et al., Proc. Natl. Acad. Sci. USA, 90:7889-93(1993).

[0141] RNA sequences encoding DEZ receptor gene protein may be directlyadministered to a patient exhibiting disease symptoms, at aconcentration sufficient to produce a level of DEZ receptor gene proteinsuch that disease symptoms are ameliorated. Patients may be treated bygene replacement therapy. One or more copies of a normal DEZ receptorgene, or a portion of the gene that directs the production of a normalDEZ receptor gene protein with DEZ receptor gene function, may beinserted into cells using vectors that include, but are not limited toadenovirus, adeno-associated virus, and retrovirus vectors, in additionto other particles that introduce DNA into cells, such as liposomes.Additionally, techniques such as those described above may be utilizedfor the introduction of normal DEZ receptor gene sequences into humancells.

[0142] Cells, preferably, autologous cells, containing normal DEZreceptor gene expressing gene sequences may then be introduced orreintroduced into the patient at positions that allow for theamelioration of disease symptoms.

[0143] Pharmaceutical Compositions, Effective Dosages, and Routes ofAdministration

[0144] The identified compounds that inhibit target mutant geneexpression, synthesis and/or activity can be administered to a patientat therapeutically effective doses to treat or ameliorate the disease. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms of the disease.

[0145] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds that exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0146] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0147] Pharmaceutical compositions for use in accordance with thepresent invention may be formulated in conventional manner using one ormore physiologically acceptable carriers or excipients. Thus, thecompounds and their physiologically acceptable salts and solvates may beformulated for administration by inhalation or insufflation (eitherthrough the mouth or the nose) or oral, buccal, parenteral, topical,subcutaneous, intraperitoneal, intraveneous, intrapleural, intraoccular,intraarterial, or rectal administration. It is also contemplated thatpharmaceutical compositions may be administered with other products thatpotentiate the activity of the compound and optionally, may includeother therapeutic ingredients.

[0148] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

[0149] Preparations for oral administration may be suitably formulatedto give controlled release of the active compound.

[0150] For buccal administration the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0151] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0152] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0153] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. Oralingestion is possibly the easiest method of taking any medication. Sucha route of administration, is generally simple and straightforward andis frequently the least inconvenient or unpleasant route ofadministration from the patient's point of view. However, this involvespassing the material through the stomach, which is a hostile environmentfor many materials, including proteins and other biologically activecompositions. As the acidic, hydrolytic and proteolytic environment ofthe stomach has evolved efficiently to digest proteinaceous materialsinto amino acids and oligopeptides for subsequent anabolism, it ishardly surprising that very little or any of a wide variety ofbiologically active proteinaceous material, if simply taken orally,would survive its passage through the stomach to be taken up by the bodyin the small intestine. The result, is that many proteinaceousmedicaments must be taken in through another method, such asparenterally, often by subcutaneous, intramuscular or intravenousinjection.

[0154] Pharmaceutical compositions may also include various buffers(e.g., Tris, acetate, phosphate), solubilizers (e.g., Tween,Polysorbate), carriers such as human serum albumin, preservatives(thimerosol, benzyl alcohol) and anti-oxidants such as ascorbic acid inorder to stabilize pharmaceutical activity. The stabilizing agent may bea detergent, such as tween-20, tween-80, NP-40 or Triton X-100. EBP mayalso be incorporated into particulate preparations of polymericcompounds for controlled delivery to a patient over an extended periodof time. A more extensive survey of components in pharmaceuticalcompositions is found in Remington's Pharmaceutical Sciences, 18th ed.,A. R. Gennaro, ed., Mack Publishing, Easton, Pa. (1990).

[0155] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0156] The compositions may, if desired, be presented in a pack ordispenser device that may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

[0157] Diagnostics

[0158] A variety of methods may be employed to diagnose diseaseconditions associated with the DEZ receptor gene. Specifically, reagentsmay be used, for example, for the detection of the presence of DEZreceptor gene mutations, or the detection of either over or underexpression of DEZ receptor gene mRNA.

[0159] According to the diagnostic and prognostic method of the presentinvention, alteration of the wild-type DEZ receptor gene locus isdetected. In addition, the method can be performed by detecting thewild-type DEZ receptor gene locus and confirming the lack of apredisposition or neoplasia. “Alteration of a wild-type gene”encompasses all forms of mutations including deletions, insertions andpoint mutations in the coding and noncoding regions. Deletions may be ofthe entire gene or only a portion of the gene. Point mutations mayresult in stop codons, frameshift mutations or amino acid substitutions.Somatic mutations are those that occur only in certain tissues, e.g., intumor tissue, and are not inherited in the germline. Germline mutationscan be found in any of a body's tissues and are inherited. If only asingle allele is somatically mutated, an early neoplastic state may beindicated. However, if both alleles are mutated, then a late neoplasticstate may be indicated. The finding of gene mutations thus provides bothdiagnostic and prognostic information. A DEZ receptor gene allele thatis not deleted (e.g., that found on the sister chromosome to achromosome carrying a DEZ receptor gene deletion) can be screened forother mutations, such as insertions, small deletions, and pointmutations. Mutations found in tumor tissues may be linked to decreasedexpression of the DEZ receptor gene product. However, mutations leadingto non-functional gene products may also be linked to a cancerous state.Point mutational events may occur in regulatory regions, such as in thepromoter of the gene, leading to loss or diminution of expression of themRNA. Point mutations may also abolish proper RNA processing, leading toloss of expression of the DEZ receptor gene product, or a decrease inmRNA stability or translation efficiency.

[0160] One test available for detecting mutations in a candidate locusis to directly compare genomic target sequences from cancer patientswith those from a control population. Alternatively, one could sequencemessenger RNA after amplification, e.g., by PCR, thereby eliminating thenecessity of determining the exon structure of the candidate gene.Mutations from cancer patients falling outside the coding region of theDEZ receptor gene can be detected by examining the non-coding regions,such as introns and regulatory sequences near or within the DEZ receptorgene. An early indication that mutations in noncoding regions areimportant may come from Northern blot experiments that reveal messengerRNA molecules of abnormal size or abundance in cancer patients ascompared to control individuals.

[0161] The methods described herein may be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one specificgene nucleic acid or anti-gene antibody reagent described herein, whichmay be conveniently used, e.g., in clinical settings, to diagnosepatients exhibiting disease symptoms or at risk for developing disease.

[0162] Any cell type or tissue, preferably platelets, neutrophils orlymphocytes, in which the gene is expressed may be utilized in thediagnostics described below.

[0163] DNA or RNA from the cell type or tissue to be analyzed may easilybe isolated using procedures that are well known to those in the art.Diagnostic procedures may also be performed in situ directly upon tissuesections (fixed and/or frozen) of patient tissue obtained from biopsiesor resections, such that no nucleic acid purification is necessary.Nucleic acid reagents may be used as probes and/or primers for such insitu procedures (see, for example, Nuovo, PCR In Situ Hybridization:Protocols and Applications, Raven Press, N.Y. (1992)).

[0164] Gene nucleotide sequences, either RNA or DNA, may, for example,be used in hybridization or amplification assays of biological samplesto detect disease-related gene structures and expression. Such assaysmay include, but are not limited to, Southern or Northern analyses,restriction fragment length polymorphism assays, single strandedconformational polymorphism analyses, in situ hybridization assays, andpolymerase chain reaction analyses. Such analyses may reveal bothquantitative aspects of the expression pattern of the gene, andqualitative aspects of the gene expression and/or gene composition. Thatis, such aspects may include, for example, point mutations, insertions,deletions, chromosomal rearrangements, and/or activation or inactivationof gene expression.

[0165] Preferred diagnostic methods for the detection of gene-specificnucleic acid molecules may involve for example, contacting andincubating nucleic acids, derived from the cell type or tissue beinganalyzed, with one or more labeled nucleic acid reagents underconditions favorable for the specific annealing of these reagents totheir complementary sequences within the nucleic acid molecule ofinterest. Preferably, the lengths of these nucleic acid reagents are atleast 9 to 30 nucleotides. After incubation, all non-annealed nucleicacids are removed from the nucleic acid:fingerprint molecule hybrid. Thepresence of nucleic acids from the fingerprint tissue that havehybridized, if any such molecules exist, is then detected. Using such adetection scheme, the nucleic acid from the tissue or cell type ofinterest may be immobilized, for example, to a solid support such as amembrane, or a plastic surface such as that on a microtitre plate orpolystyrene beads. In this case, after incubation, non-annealed, labelednucleic acid reagents are easily removed. Detection of the remaining,annealed, labeled nucleic acid reagents is accomplished using standardtechniques well-known to those in the art.

[0166] Alternative diagnostic methods for the detection of gene-specificnucleic acid molecules may involve their amplification, e.g., by PCR(the experimental embodiment set forth in Mullis U.S. Pat. No. 4,683,202(1987)), ligase chain reaction (Barany, Proc. Natl. Acad. Sci. USA,88:189-93 (1991)), self sustained sequence replication (Guatelli, etal., Proc. Natl. Acad. Sci. USA, 87:1874-78 (1990)), transcriptionalamplification system (Kwoh, et al., Proc. Natl. Acad. Sci. USA,86:1173-77 (1989)), Q-Beta Replicase (Lizardi et al., Bio/Technology,6:1197 (1988)), or any other nucleic acid amplification method, followedby the detection of the amplified molecules using techniques well knownto those of skill in the art. These detection schemes are especiallyuseful for the detection of nucleic acid molecules if such molecules arepresent in very low numbers.

[0167] In one embodiment of such a detection scheme, a cDNA molecule isobtained from an RNA molecule of interest (e.g., by reversetranscription of the RNA molecule into cDNA). Cell types or tissues fromwhich such RNA may be isolated include any tissue in which wild typefingerprint gene is known to be expressed, including, but not limited,to platelets, neutrophils and lymphocytes. A sequence within the cDNA isthen used as the template for a nucleic acid amplification reaction,such as a PCR amplification reaction, or the like. The nucleic acidreagents used as synthesis initiation reagents (e.g., primers) in thereverse transcription and nucleic acid amplification steps of thismethod may be chosen from among the gene nucleic acid reagents describedherein. The preferred lengths of such nucleic acid reagents are at least15-30 nucleotides. For detection of the amplified product, the nucleicacid amplification may be performed using radioactively ornon-radioactively labeled nucleotides. Alternatively, enough amplifiedproduct may be made such that the product may be visualized by standardethidium bromide staining or by utilizing any other suitable nucleicacid staining method.

[0168] Antibodies directed against wild type or mutant gene peptides mayalso be used as disease diagnostics and prognostics. Such diagnosticmethods, may be used to detect abnormalities in the level of geneprotein expression, or abnormalities in the structure and/or tissue,cellular, or subcellular location of fingerprint gene protein.Structural differences may include, for example, differences in thesize, electronegativity, or antigenicity of the mutant fingerprint geneprotein relative to the normal fingerprint gene protein.

[0169] Protein from the tissue or cell type to be analyzed may easily bedetected or isolated using techniques that are well known to those ofskill in the art, including but not limited to western blot analysis.For a detailed explanation of methods for carrying out western blotanalysis, see Sambrook, et al. (1989) supra, at Chapter 18. The proteindetection and isolation methods employed herein may also be such asthose described in Harlow and Lane, for example, (Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1988)).

[0170] Preferred diagnostic methods for the detection of wild type ormutant gene peptide molecules may involve, for example, immunoassayswherein fingerprint gene peptides are detected by their interaction withan anti-fingerprint gene-specific peptide antibody.

[0171] For example, antibodies, or fragments of antibodies useful in thepresent invention may be used to quantitatively or qualitatively detectthe presence of wild type or mutant gene peptides. This can beaccomplished, for example, by immunofluorescence techniques employing afluorescently labeled antibody (see below) coupled with lightmicroscopic, flow cytometric, or fluorimetric detection. Such techniquesare especially preferred if the fingerprint gene peptides are expressedon the cell surface.

[0172] The antibodies (or fragments thereof) useful in the presentinvention may, additionally, be employed histologically, as inimmunofluorescence or immunoelectron microscopy, for in situ detectionof fingerprint gene peptides. In situ detection may be accomplished byremoving a histological specimen from a patient, and applying thereto alabeled antibody of the present invention. The antibody (or fragment) ispreferably applied by overlaying the labeled antibody (or fragment) ontoa biological sample. Through the use of such a procedure, it is possibleto determine not only the presence of the fingerprint gene peptides, butalso their distribution in the examined tissue. Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

[0173] Immunoassays for wild type, mutant, or expanded fingerprint genepeptides typically comprise incubating a biological sample, such as abiological fluid, a tissue extract, freshly harvested cells, or cellsthat have been incubated in tissue culture, in the presence of adetectably labeled antibody capable of identifying fingerprint genepeptides, and detecting the bound antibody by any of a number oftechniques well known in the art.

[0174] The biological sample may be brought in contact with andimmobilized onto a solid phase support or carrier such asnitrocellulose, or other solid support that is capable of immobilizingcells, cell particles or soluble proteins. The support may then bewashed with suitable buffers followed by treatment with the detectablylabeled gene-specific antibody. The solid phase support may then bewashed with the buffer a second time to remove unbound antibody. Theamount of bound label on solid support may then be detected byconventional means.

[0175] The terms “solid phase support or carrier” are intended toencompass any support capable of binding an antigen or an antibody.Well-known supports or carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, gabbros, and magnetite. The natureof the carrier can be either soluble to some extent or insoluble for thepurposes of the present invention. The support material may havevirtually any possible structural configuration so long as the coupledmolecule is capable of binding to an antigen or antibody. Thus, thesupport configuration may be spherical, as in a bead, or cylindrical, asin the inside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, test strip, etc.Preferred supports include polystyrene beads. Those skilled in the artwill know many other suitable carriers for binding antibody or antigen,or will be able to ascertain the same by use of routine experimentation.

[0176] The binding activity of a given lot of anti-wild type or -mutantfingerprint gene peptide antibody may be determined according to wellknown methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

[0177] One of the ways in which the gene peptide-specific antibody canbe detectably labeled is by linking the same to an enzyme and using itin an enzyme immunoassay (EIA) (Voller, Ric Clin Lab, 8:289-98 (1978)[“The Enzyme Linked Immunosorbent Assay (ELISA)”, Diagnostic Horizons2:1-7, 1978, Microbiological Associates Quarterly Publication,Walkersville, Md.]; Voller, et al., J. Clin. Pathol., 31:507-20 (1978);Butler, Meth. Enzymol., 73:482-523 (1981); Maggio (ed.), EnzymeImmunoassay, CRC Press, Boca Raton, Fla. (1980); Ishikawa, et al.,(eds.) Enzyme Immunoassay, Igaku-Shoin, Tokyo (1981)). The enzyme thatis bound to the antibody will react with an appropriate substrate,preferably a chromogenic substrate, in such a manner as to produce achemical moiety that can be detected, for example, byspectrophotometric, fluorimetric or by visual means. Enzymes that can beused to detectably label the antibody include, but are not limited to,malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate,dehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. The detection can be accomplishedby calorimetric methods that employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

[0178] Detection may also be accomplished using any of a variety ofother immunoassays. For example, by radioactively labeling theantibodies or antibody fragments, it is possible to detect fingerprintgene wild type, mutant, or expanded peptides through the use of aradioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March, 1986). The radioactive isotopecan be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

[0179] It is also possible to label the antibody with a fluorescentcompound. When the fluorescently labeled antibody is exposed to light ofthe proper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

[0180] The antibody can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriaminepentacetic acid (DTPA) orethylenediamine-tetraacetic acid (EDTA).

[0181] The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

[0182] Likewise, a bioluminescent compound may be used to label theantibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

[0183] Throughout this application, various publications, patents andpublished patent applications are referred to by an identifyingcitation. The disclosures of these publications, patents and publishedpatent specifications referenced in this application are herebyincorporated by reference into the present disclosure to more fullydescribe the state of the art to which this invention pertains.

[0184] The following examples are intended only to illustrate thepresent invention and should in no way be construed as limiting thesubject invention.

EXAMPLES Example 1 Generation and Analysis of Mice Comprising GPCR GeneDisruptions

[0185] Targeting Construct for DEZ Receptor Gene. To investigate therole of genes encoding GPCRs, particularly, DEZ receptor genes,disruptions in genes comprising the sequence set forth in SEQ ID NO:1were produced by homologous recombination. More particularly, as shownin FIGS. 2A-2B, a specific targeting construct having the ability todisrupt or modify genes, specifically comprising SEQ ID NO:1 was createdusing as the targeting arms (homologous sequences) in the construct, thesequences identified herein as SEQ ID NO:2 and SEQ ID NO:3.

[0186] Transgenic Mice. The targeting construct was introduced into EScells by electroporation and chimeric mice were generated. The ES cellswere derived from 129/Sv-+P+Mgf-SLJ/J mouse substrain. F1 mice weregenerated by breeding with C57BL/6 females. The resultant F1N0heterozygotes were intercrossed to produce F2N0 homozygotes, or werebackcrossed to C57BL/6 mice to generate F1N1 heterozygotes. F2N1homozygous mutant mice were produced by intercrossing F1N1 heterozygousmales and females.

[0187] Phenotypic Analysis. The transgenic mice were analyzed forphenotypic changes. When compared to age- and gender-matched wild-typecontrol mice, homozygous mutant mice exhibited decreased agility orcoordination, as characterized by decreased performance (i.e., decreasedlatency) on an accelerating rotarod, i.e., the homozygous mutants fellfrom the rod at a lower speed than their wild-type counterparts, whichincreased their latency to fall across three consecutive trials (seeTable 1). TABLE 1 ROTAROD FALL SPEED Rotarod F N Speed Std. FamilySubFamily Gene Name Genotype Generation Generation Trial (rpm) Err.Count GPCR Orphan GPCR GPCR Dez +/+ 2 0 Trial 1 6.47 0.67 12 GPCR OrphanGPCR GPCR Dez +/+ 2 0 Trial 2 7.88 1.17 12 GPCR Orphan GPCR GPCR Dez +/+2 0 Trial 3 8.71 1.51 12 GPCR Orphan GPCR GPCR Dez +/+ 2 1 Trial 1 7.631.16 10 GPCR Orphan GPCR GPCR Dez +/+ 2 1 Trial 2 10.50 1.43 10 GPCROrphan GPCR GPCR Dez +/+ 2 1 Trial 3 11.69 0.90 10 GPCR Orphan GPCR GPCRDez −/− 2 0 Trial 1 5.90 0.71 12 GPCR Orphan GPCR GPCR Dez −/− 2 0 Trial2 5.99 0.72 12 GPCR Orphan GPCR GPCR Dez −/− 2 0 Trial 3 7.00 0.88 12GPCR Orphan GPCR GPCR Dez −/− 2 1 Trial 1 7.21 0.51 10 GPCR Orphan GPCRGPCR Dez −/− 2 1 Trial 2 7.99 0.82 10 GPCR Orphan GPCR GPCR Dez −/− 2 1Trial 3 8.15 0.87 10

[0188] As is apparent to one of skill in the art, various modificationsof the above embodiments can be made without departing from the spiritand scope of this invention. These modifications and variations arewithin the scope of this invention.

We claim:
 1. A targeting construct comprising: (a) a firstpolynucleotide sequence homologous to a DEZ receptor gene; (b) a secondpolynucleotide sequence homologous to the DEZ receptor gene; and (c) aselectable marker.
 2. The targeting construct of claim 1, wherein thetargeting construct further comprises a screening marker.
 3. A method ofproducing a targeting construct, the method comprising: (a) providing afirst polynucleotide sequence homologous to a DEZ receptor gene; (b)providing a second polynucleotide sequence homologous to the DEZreceptor gene; (c) providing a selectable marker; and (d) inserting thefirst sequence, second sequence, and selectable marker into a vector, toproduce the targeting construct.
 4. A method of producing a targetingconstruct, the method comprising: (a) providing a polynucleotidecomprising a first sequence homologous to a first region of a DEZreceptor gene and a second sequence homologous to a second region of aDEZ receptor gene; and (b) inserting a positive selection marker betweenthe first and second sequences to form the targeting construct.
 5. Acell comprising a disruption in a DEZ receptor gene.
 6. The cell ofclaim 5, wherein the cell is a murine cell.
 7. The cell of claim 6,wherein the murine cell is an embryonic stem cell.
 8. A non-humantransgenic animal comprising a disruption in a DEZ receptor gene.
 9. Acell derived from the non-human transgenic animal of claim
 8. 10. Amethod of producing a transgenic mouse comprising a disruption in a DEZreceptor gene, the method comprising: (a) introducing the targetingconstruct of claim 1 into a cell; (b) introducing the cell into ablastocyst; (c) implanting the resulting blastocyst into apseudopregnant mouse, wherein said pseudopregnant mouse gives birth to achimeric mouse; and (d) breeding the chimeric mouse to produce thetransgenic mouse.
 11. A method of identifying an agent that modulatesthe expression of a DEZ receptor, the method comprising: (a) providing anon-human transgenic animal comprising a disruption in a DEZ receptorgene; (b) administering an agent to the non-human transgenic animal; and(c) determining whether the expression of DEZ receptor in the non-humantransgenic animal is modulated.
 12. A method of identifying an agentthat modulates the function of a DEZ receptor, the method comprising:(a) providing a non-human transgenic animal comprising a disruption in aDEZ receptor gene; (b) administering an agent to the non-humantransgenic animal; and (c) determining whether the function of thedisrupted DEZ receptor gene in the non-human transgenic animal ismodulated.
 13. A method of identifying an agent that modulates theexpression of DEZ receptor, the method comprising: (a) providing a cellcomprising a disruption in a DEZ receptor gene; (b) contacting the cellwith an agent; and (c) determining whether expression of the DEZreceptor is modulated.
 14. A method of identifying an agent thatmodulates the function of a DEZ receptor gene, the method comprising:(a) providing a cell comprising a disruption in a DEZ receptor gene; (b)contacting the cell with an agent; and (c) determining whether thefunction of the DEZ receptor gene is modulated.
 15. The method of claim13 or claim 14, wherein the cell is derived from the non-humantransgenic animal of claim
 8. 16. An agent identified by the method ofclaim 11, claim 12, claim 13, or claim
 14. 17. A transgenic mousecomprising a homozygous disruption in a gene comprising SEQ ID NO:1, ora homolog thereof.
 18. The transgenic mouse of claim 17, wherein thetransgenic mouse exhibits decreased agility or coordination relative toa wild-type control mouse.
 19. The transgenic mouse of claim 18, whereinthe decreased agility or coordination is characterized by decreasedlatency in an accelerating rotarod test.
 20. Phenotypic data associatedwith the transgenic mouse of claim 17, wherein the phenotypic data is ina database